Papilloma pseudo-virus and preparation

ABSTRACT

The invention involves a papilloma pseudo-virus that can induce immune response after oral intake as well as its preparation. It characterizes that: the HPV or BPV pseudo-virus made by disrupting HPV-VLP or BPV-VLP, mixing them with plasmids (plasmids or DNA vaccine), reassembled into pseudo-virus (VLPs with plasmids inside). The pseudo-virus can be orally given, entering mucosa and systemic lymphoid tissues of human body, activate immune system and serve as treating and preventing agent. The pseudo-virus can induce stronger immune response than DNA vaccines. Additionally, the pseudo-virus can be applied in gene therapy by bringing the therapeutic genes into lymphoid tissues in human body.

TECHNICAL FIELD

[0001] This patent involves a papiloma pseudo-virus and its preparation. The papilloma pseudo-virus can induce immune responses when given orally. This pseudo-virus can be used as vaccine to treat and prevent mucosal pathogen infections or mucosal tumors. Additionally it can be applied in gene therapy.

TECHNICAL BACKGROUND

[0002] It is well known that pathogens and immune system deficiency are the major causes of various diseases. Human body is frequently invaded by pathogens and damaged by inner tumors. Therefore, immunity is necessary in preventing human body from various infections and inner pathological changes. Many vaccines currently used induce specific immune response through subcutaneous and intramuscular injections and help body survive the disease. However, the injection can only induce systemic immune responses but not mucosal immune responses. The injected vaccine fails to treat and prevent those pathogens transmitted through mucosa. Actually many diseases are transmitted through mucosa, for example, HIV.

DESCRIPTION OF THE INVENTION

[0003] The objective of this patent is to work out a pseudo-virus which is similar to the virus but without the capacity of causing disease. The gene of DNA which has therapeutic and prevention efficiency was packaged into virus-like particles (VLPs). The pseudo-virus is taken orally or from other mucosa and then absorbed by mucosa and lymph tissues, inducing the immune response to serve as treatment and prevention.

[0004] The pseudo-virus introduced in the patent is made by disrupting HPV-VLP or BPV-VLP, mixing them with plasmids. The virus will be reassembled with plasmids inside the VLPs. The pseudo-virus contains VLPs but without viral contents-DNA. If the DNA vaccine is put into VLPs, the vaccine can enter body by taking orally. The vaccine in the patent does not contain DNA of papilloma virus. The DNA vaccine will induce systemic immune response by taking through subcutaneous or intramuscular injections. In the other word, the pseudo-virus in the invention is VLPs that contain the DNA vaccine. It is prepared that by following steps:

[0005] 1. HPV-VLPs or BPV-VLPs are mixed with disruption buffer by 1:1 ratio in vol., incubated 60 min, at room temperature; disruption buffer: ethylene glycol bis(2-aminoethylether) tetraacetic acid (EGTA) 20 mM, dithiothreitol (DTT) 40 mM, sodium chloride (NaCl) 300 mM, Tris-hydrochloric acid (Tris-HCl)(pH 8.0) 100 mM;

[0006] 2. Add plasmids in {fraction (1/10)} of vol, 0.5-1.0 microgram/microliter;

[0007] 3. Put in stop buffer progressively. The stop buffer; calcium chloride (CaCl₂) 25 mM, dimethyl sulfoxide (DMSO) 20% (total stop buffer in vol.)

[0008] 4. Incubate the mixture at 4 centigrade for 4 to 12 hours.

EMBODIMEMT Example 1

[0009] First, the VLP was mixed with disruption buffer by 1:1 proportion in vol., and then incubate at room temperature for 60 min. Disruption buffer: ethylene glycol bis(2-aminoethylether) tetraacetic acid (EGTA) 20 mM, dithiothreitol (DTT) 40 mM, sodium chloride (NaCl) 300 mM, Tris-hydrochloric acid (Tris-HCl)(pH 8.0) 100 mM. Secondly, the PCl-GLP-LCMV plasmids were put in 0.5 microgram/microliter by ratio of {fraction (1/10)} in vol. Next, progressively same volume stop buffer was put, incubate at 4 centigrade overnight. Stop buffer: calcium chloride (CaCl₂) 25 mM, dimethyl sulfoxide (DMSO) 20% (in vol.). The pseudo-virus was subcutaneously injected to C57BL6 mice. Meanwhile, the unpackaged plasmids were injected directly into control mice. It found that the pseudo-virus induced more CTLs than plasmids did by using Cr51 release assay or gamma interferon Elispot. It comes the conclusion that the pseudo-virus is more efficient than DNA vaccines in inducing immune response.

Example 2

[0010] First, the VLPs were mixed with disruption buffer by 1:1 proportion in vol., and then incubate at room temperature for 60 min. Disruption buffer. EGTA 20 mM, DTT 40 mM, NaCl 300 mM, Tris-hydrochloric acid (Tris-HCl) (pH 8.0) 100 mM. Secondly, plasmid expressing GLP (Green lantern protein) was added in 0.5 microgram/microliter by ratio of {fraction (1/10)} in vol. Next, same volume stop buffer was added, incubate at 4 centigrade overnight. Stop buffer: CaCl₂ 25 mM, DMSO 20% (in vol.). The pseudo-viruses were orally administered into mice, the expression of GLP was examined. It found that GLP in intestinal mucosa, mesenteric lymph nodes, and spleen. It is demonstrated that the pseudo-virus can carry genes to intestinal mucosa and entire immune system. Therefore, it can be used in gene therapy. GLP has not been in mice taking plasmids orally.

Example 3

[0011] By the same method as example 1, pseudo-virus expressing HPV16E7 was prepared and given to mice orally. Pseudo-virus induced specific mucosal and systemic CTLs to E7, but plasmids could not do from oral immunization. Therefore, pseudo-virus can be used to induce mucosal and systemic immune response.

Example 4

[0012] By the same method as example 1 and 2, pseudo-virus expressing HPV16E7 by using HPV and BPV VLPs respectively was prepared. First, HPV pseudo-virus was given to mice orally and then challenged mice with BPV pseudo-virus. It was observed that HPV pseudo-virus prevented mice form challenging with BPV pseudo-virus. Therefore, HPV pseudo-virus can provide immunity.

Example 5

[0013] By the same method as described in example 1 and 2, the pseudo-virus expressing interleukin 2 (IL-2) was prepared. In aged mice, vaccines do not induce effective immune responses. It found that the pseudo-virus expressing IL-2 restored the capacity of aged mice to respond to vaccination.

Industrial Application

[0014] The pseudo-virus prepared the gene into plasmids and packaging the plasmid into virus-like particles, which will be applied in gene therapy, is not pathogenic. The pseudo-virus brings the DNA vaccine orally into intestinal mucosa, systematic lymph tissues and mucosal epithelium. In addition, the pseudo-virus can serve as a vaccine. Inserting the antigen gene into plasmids, it can make the pseudo-virus that can induce protective immune response after oral immunization. It differs from many current used vaccines that can only be applied in subcutaneous or intramuscular injections rather than oral application and induce only the systemic immune response rather than the mucosal immune response. Owing to the fact that diseases are transmitted through mucosa, only the pseudo-virus will induce mucosal immune response to treat many pathogens and prevent them from infecting human body through mucosa. The pathogens invading human body through mucosa include germs, such as salmonella, and virus, such as HIV (Human Immunodeficiency Virus). The pseudo-virus can also be applied to deal with tumors especially mucosal tumors, for instance, colon tumor, by inducing anti-tumor immune response. The pseudo-virus can induce stronger immune response than DNA vaccines. 

What is claimed is:
 1. A Papilloma pseudo-virus is prepared by disruption of HPV-VLP or BPV-VLP (or other animals), and then mix them with plasmids, reassembled into pseudo-virus (VLPs with plasmids inside).
 2. An instruction of preparation of a papilloma pseudo-virus which character is that: (1) HPV-VLP or BPV-VLP is mixed with disruption buffer by 1:1 ratio in vol., incubated 60 min, at room temperature; disruption buffer: EGTA 20 mM, DTT 40 mM, NaCl 300 mM, Tris-hydrochloric acid (Tris-HCl) (pH 8.0) 100 mM. (2) Add in plasmids in {fraction (1/10)} ratio, concentration 0.5-1.0 microgram/microliter; (3) Add stop buffer gradually. The stop buffer: CaCl₂ 25 mM, DMSO 20% (in vol.) (4) Incubate the mixture at 4 centigrade from 4 to 12 hours.
 3. According to the preparation instruction described in claim 2, the pseudo-virus which is prepared by inserting some gene of pathogens into plasmids can be used to prevent and to treat any diseases.
 4. According to the preparation instruction described in claim 2, the pseudo-virus which is prepared by inserting therapeutic genes into plasmids can be used to treat any diseases, any deficiency caused by aging.
 5. According to the preparation instruction described in claim 2, HPV-VLP or BPV-VLP are mixed with disruption buffer by 1:1 ratio in vol.
 6. According to the preparation instruction described in claim 2, the total volume of stop buffer should equal that total volume of intermediate products from step one and step two. 